Dominant Negative Forms of Akt (Protein Kinase B) and Atypical Protein Kinase Cλ Do Not Prevent Insulin Inhibition of Phosphoenolpyruvate Carboxykinase Gene Transcription
1999; Elsevier BV; Volume: 274; Issue: 30 Linguagem: Inglês
10.1074/jbc.274.30.21305
ISSN1083-351X
AutoresKo Kotani, Wataru Ogawa, Yasuhisa Hino, Tadahiro Kitamura, Hikaru Ueno, Wataru Sano, Calum Sutherland, Daryl K. Granner, Masato Kasuga,
Tópico(s)Metabolism, Diabetes, and Cancer
ResumoTranscriptional regulation of phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme in hepatic gluconeogenesis, by insulin was investigated with the use of adenovirus vectors encoding various mutant signaling proteins. Insulin inhibited transcription induced by dexamethasone and cAMP of a chloramphenicol acetyltransferase (CAT) reporter gene fused with the PEPCK promoter sequence in HL1C cells stably transfected with this construct. A dominant negative mutant of phosphoinositide (PI) 3-kinase blocked insulin inhibition of transcription of the PEPCK-CAT fusion gene, whereas a constitutively active mutant of PI 3-kinase mimicked the effect of insulin. Although a constitutively active mutant of Akt (protein kinase B) inhibited PEPCK-CAT gene transcription induced by dexamethasone and cAMP, a mutant Akt (Akt-AA) in which the phosphorylation sites targeted by insulin are replaced by alanine did not affect the ability of insulin to inhibit transcription of the fusion gene. Akt-AA almost completely inhibited insulin-induced activation of both endogenous and recombinant Akt in HL1C cells. Furthermore, neither a kinase-defective mutant protein kinase Cλ (PKCλ), which blocked insulin-induced activation of endogenous PKCλ, nor a dominant negative mutant of the small GTPase Rac prevented inhibition of PEPCK-CAT gene transcription by insulin. These data suggest that phosphoinositide 3-kinase is important for insulin-induced inhibition of PEPCK gene transcription and that a downstream effector of phosphoinositide 3-kinase distinct from Akt, PKCλ, and Rac may exist for mediating the effect of insulin. Transcriptional regulation of phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme in hepatic gluconeogenesis, by insulin was investigated with the use of adenovirus vectors encoding various mutant signaling proteins. Insulin inhibited transcription induced by dexamethasone and cAMP of a chloramphenicol acetyltransferase (CAT) reporter gene fused with the PEPCK promoter sequence in HL1C cells stably transfected with this construct. A dominant negative mutant of phosphoinositide (PI) 3-kinase blocked insulin inhibition of transcription of the PEPCK-CAT fusion gene, whereas a constitutively active mutant of PI 3-kinase mimicked the effect of insulin. Although a constitutively active mutant of Akt (protein kinase B) inhibited PEPCK-CAT gene transcription induced by dexamethasone and cAMP, a mutant Akt (Akt-AA) in which the phosphorylation sites targeted by insulin are replaced by alanine did not affect the ability of insulin to inhibit transcription of the fusion gene. Akt-AA almost completely inhibited insulin-induced activation of both endogenous and recombinant Akt in HL1C cells. Furthermore, neither a kinase-defective mutant protein kinase Cλ (PKCλ), which blocked insulin-induced activation of endogenous PKCλ, nor a dominant negative mutant of the small GTPase Rac prevented inhibition of PEPCK-CAT gene transcription by insulin. These data suggest that phosphoinositide 3-kinase is important for insulin-induced inhibition of PEPCK gene transcription and that a downstream effector of phosphoinositide 3-kinase distinct from Akt, PKCλ, and Rac may exist for mediating the effect of insulin. The primary role of insulin is to control the plasma glucose concentration by stimulating glucose transport into muscle and adipose cells as well as by reducing glucose output from the liver (1Kruszynska Y.T. Olefsky J.M. J. Invest. Med. 1996; 44: 413-428PubMed Google Scholar). These actions of insulin are mediated by activation of effectors, such as glucose transporters and glycogen synthase, or by regulation of the amount of specific protein participants in metabolic pathways (1Kruszynska Y.T. Olefsky J.M. J. Invest. Med. 1996; 44: 413-428PubMed Google Scholar, 2O'Brien R.M. Granner D.K. Biochem. J. 1991; 278: 609-619Crossref PubMed Scopus (246) Google Scholar, 3White M.F. Diabetologia. 1997; 40 Suppl. 2: 2-17Crossref PubMed Scopus (460) Google Scholar, 4Holman G.D. Kasuga M. Diabetologia. 1997; 40: 991-1003Crossref PubMed Scopus (187) Google Scholar). Phosphoenolpyruvate carboxykinase (PEPCK), 1The abbreviations used are: PEPCK, phosphoenolpyruvate carboxykinase; PI, phosphoinositide; PKC, protein kinase C; CAT, chloramphenicol acetyltransferase; HA, hemagglutinin; MOI, multiplicity of infection; pfu, plaque-forming unit1The abbreviations used are: PEPCK, phosphoenolpyruvate carboxykinase; PI, phosphoinositide; PKC, protein kinase C; CAT, chloramphenicol acetyltransferase; HA, hemagglutinin; MOI, multiplicity of infection; pfu, plaque-forming unit a rate controlling enzyme of gluconeogenesis, is one such protein whose expression is regulated by insulin (2O'Brien R.M. Granner D.K. Biochem. J. 1991; 278: 609-619Crossref PubMed Scopus (246) Google Scholar). Insulin inhibits gluconeogenesis in the liver; thus, in the absence of this effect of insulin, as in diabetes mellitus or long term starvation, gluconeogenesis is increased (1Kruszynska Y.T. Olefsky J.M. J. Invest. Med. 1996; 44: 413-428PubMed Google Scholar). Transcription of the PEPCK gene is increased by various hormonal agents including glucocorticoids and glucagon (or its second messenger, cAMP), and insulin inhibits PEPCK gene transcription induced by these stimuli (2O'Brien R.M. Granner D.K. Biochem. J. 1991; 278: 609-619Crossref PubMed Scopus (246) Google Scholar). Given that PEPCK is not known to be subject to allosteric regulation, the inhibition of gluconeogenesis by insulin in vivo is probably due to the insulin-induced decrease in the amount of PEPCK protein. Moreover, the observations that PEPCK gene expression in the liver is increased in several animal models of diabetes (5Hofmann C. Lorenz K. Williams D. Palazuk B.J. Colca J.R. Metabolism. 1995; 44: 384-389Abstract Full Text PDF PubMed Scopus (25) Google Scholar) and that transgenic animals overexpressing the PEPCK gene develop a diabetic phenotype (6Valera A. Pujol A. Pelegrin M. Bosch F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9151-9154Crossref PubMed Scopus (252) Google Scholar, 7Rosella G. Zajac J.D. Baker L. Kaczmarczyk S.J. Andrikopoulos S. Adams T.E. Proietto J. Mol. Endocrinol. 1995; 9: 1396-1404PubMed Google Scholar) also indicate the importance of this enzyme in glucose homeostasis in vivo.Despite the recent progress in our knowledge of insulin signal transduction, the mechanism by which PEPCK gene transcription is regulated remains unclear. The Ras and mitogen-activated protein kinase signaling cascade contributes to the regulation of the expression of various genes by insulin (8Davis R.J. Mol. Reprod. Dev. 1995; 42: 459-467Crossref PubMed Scopus (382) Google Scholar). A constitutively active mutant of Ras was shown to inhibit transcription of the PEPCK gene induced by cAMP-dependent protein kinase (9Agati J.M. Yeagley D. Quinn P.G. J. Biol. Chem. 1998; 273: 18751-18759Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). In contrast, constitutively active mutants of Ras or of Raf, an immediate downstream effector of Ras, did not inhibit PEPCK gene transcription induced by dexamethasone and a cAMP analog (10Gabbay R.A. Sutherland C. Gnudi L. Kahn B.B. O'Brien R.M. Granner D.K. Flier J.S. J. Biol. Chem. 1996; 271: 1890-1897Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 11Sutherland C. Waltner-Law M. Gnudi L. Kahn B.B. Granner D.K. J. Biol. Chem. 1998; 273: 3198-3204Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Despite the apparently discrepant results obtained with these constitutively active mutants, the observation that either a dominant negative mutant of Ras or a pharmacological inhibitor that blocks farnesylation of Ras, an important step in the activation of this GTPase, did not prevent insulin inhibition of PEPCK gene transcription (9Agati J.M. Yeagley D. Quinn P.G. J. Biol. Chem. 1998; 273: 18751-18759Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 10Gabbay R.A. Sutherland C. Gnudi L. Kahn B.B. O'Brien R.M. Granner D.K. Flier J.S. J. Biol. Chem. 1996; 271: 1890-1897Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 11Sutherland C. Waltner-Law M. Gnudi L. Kahn B.B. Granner D.K. J. Biol. Chem. 1998; 273: 3198-3204Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) suggests that Ras is not required for this effect of insulin.The role of PI 3-kinase, which is involved in a number of the metabolic actions of insulin (12Shepherd P.R. Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (835) Google Scholar), on PEPCK gene transcription has also been explored. Yang and Dickson (13Yang S.H. Dickson A.J. Biochem. J. 1995; 310: 375-378Crossref PubMed Scopus (16) Google Scholar) showed that insulin-induced inhibition of PEPCK gene expression was not affected by a pharmacological blocker of PI 3-kinase, to which various metabolic actions of insulin are sensitive (12Shepherd P.R. Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (835) Google Scholar). However, a previous observation by Sutherland et al. (14Sutherland C. O'Brien R.M. Granner D.K. J. Biol. Chem. 1995; 270: 15501-15506Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), subsequently confirmed by others (10Gabbay R.A. Sutherland C. Gnudi L. Kahn B.B. O'Brien R.M. Granner D.K. Flier J.S. J. Biol. Chem. 1996; 271: 1890-1897Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 15Liao J. Barthel A. Nakatani K. Roth R.A. J. Biol. Chem. 1998; 273: 27320-27324Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), demonstrated that the inhibitory effect of insulin on PEPCK gene transcription is dependent on PI 3-kinase. The role of Akt (also known as protein kinase B), an immediate downstream effector of PI 3-kinase, in regulation of PEPCK gene transcription is also controversial. A kinase-deficient mutant of Akt, in which ligand-induced phosphorylation sites were replaced for alanine, prevents the inhibition of PEPCK transcription induced by insulin (15Liao J. Barthel A. Nakatani K. Roth R.A. J. Biol. Chem. 1998; 273: 27320-27324Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). By contrast, Agati et al. (9Agati J.M. Yeagley D. Quinn P.G. J. Biol. Chem. 1998; 273: 18751-18759Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) showed that kinase-deficient Akt variants containing an amino acid substitution in the kinase domain or in the PH domain did not inhibit insulin-induced repression of PEPCK transcription.In this study, we have further explored the role of PI 3-kinase in the inhibition of PEPCK gene transcription by insulin, by using dominant negative and constitutively active mutants of the enzyme. Moreover, we have investigated the roles of several downstream targets of PI 3-kinase, including Akt, atypical protein kinase C (PKC), and the small GTPase Rac, in this action of insulin.DISCUSSIONPharmacological inhibitors of PI 3-kinase, such as wortmannin and LY294002, are useful tools for exploring insulin signal transduction. Various metabolic actions of insulin, including stimulation of glucose transport, glycogen synthase, amino acid transport, and general protein synthesis, are sensitive to these inhibitors (3White M.F. Diabetologia. 1997; 40 Suppl. 2: 2-17Crossref PubMed Scopus (460) Google Scholar, 4Holman G.D. Kasuga M. Diabetologia. 1997; 40: 991-1003Crossref PubMed Scopus (187) Google Scholar, 12Shepherd P.R. Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (835) Google Scholar). These compounds also prevent the repression of PEPCK gene transcription by insulin (10Gabbay R.A. Sutherland C. Gnudi L. Kahn B.B. O'Brien R.M. Granner D.K. Flier J.S. J. Biol. Chem. 1996; 271: 1890-1897Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 14Sutherland C. O'Brien R.M. Granner D.K. J. Biol. Chem. 1995; 270: 15501-15506Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 15Liao J. Barthel A. Nakatani K. Roth R.A. J. Biol. Chem. 1998; 273: 27320-27324Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), but like most inhibitors, their effects may not be completely specific, and a number of approaches should be used in assessing the role of a particular signal transduction step in a given pathway. Accordingly, we here show that a dominant negative mutant of PI 3-kinase, Δp85, blocked this effect of insulin. Moreover, a constitutively active form of PI 3-kinase (Myr-p110) mimicked the effect of insulin on PEPCK gene expression. These data indicate that insulin-induced inhibition of PEPCK gene transcription is mediated by PI 3-kinase.Akt variants in which the Akt sequence is ligated to either a myristoylation signal sequence or a viral Gag protein exhibit kinase activity that is greater than that of the wild-type enzyme (26Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1871) Google Scholar, 27Kohn A.D. Takeuchi F. Roth R.A. J. Biol. Chem. 1996; 271: 21920-21926Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar). The expression of such mutants promotes glucose transport, general protein synthesis, glycogen synthase activity, p70 S6 kinase activity, and phosphorylation of 4E-BP1 (also known as PHAS1) (26Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1871) Google Scholar, 27Kohn A.D. Takeuchi F. Roth R.A. J. Biol. Chem. 1996; 271: 21920-21926Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, 28Ueki K. Yamamoto-Honda R. Kaburagi Y. Yamauchi T. Tobe K. Burgering B.M. Coffer P.J. Komuro I. Akanuma Y. Yazaki Y. Kadowaki T. J. Biol. Chem. 1998; 273: 5315-5322Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar), all of which are stimulated by insulin in a PI 3-kinase-dependent manner (12Shepherd P.R. Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (835) Google Scholar). We have now shown that stimulation of PEPCK gene transcription by dexamethasone-cAMP was markedly attenuated in HL1C cells that express a constitutively active mutant of Akt (Myr-Akt), an observation consistent with the results of a previous study (15Liao J. Barthel A. Nakatani K. Roth R.A. J. Biol. Chem. 1998; 273: 27320-27324Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). However, whereas the expression of Akt-AA inhibited the insulin-induced activation of Akt as well as insulin-induced stimulation of protein synthesis (17Kitamura T. Ogawa W. Sakaue H. Hino Y. Kuroda S. Takata M. Matsumoto M. Maeda T. Konishi H. Kikkawa U. Kasuga M. Mol. Cell. Biol. 1998; 18: 3708-3717Crossref PubMed Scopus (295) Google Scholar), activation of glycogen synthase (29Takata M. Ogawa W. Kitamura T. Hino Y. Kuroda S. Kotani K. Klip A. Gingras A.C. Sonenberg N. Kasuga J. Biol. Chem. 1999; 274: 20611-20618Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), phosphorylation of PHAS1 (29Takata M. Ogawa W. Kitamura T. Hino Y. Kuroda S. Kotani K. Klip A. Gingras A.C. Sonenberg N. Kasuga J. Biol. Chem. 1999; 274: 20611-20618Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), and phos- phorylation and activation of the 3B isoform of cAMP phosphodiesterase (PDE3B), 2Kitamura, T., Ogawa, W., Hino. Y., and Kausga, M., submitted for publication. this mutant did not affect the inhibition of PEPCK gene transcription by insulin.It is possible that Akt-AA did not completely prevent signal transmission through endogenous Akt and that the remaining low level of Akt activity was sufficient to inhibit PEPCK gene transcription to a substantial extent. However, this possibility is unlikely because both insulin-induced Akt activation (data not shown) and PEPCK gene transcription were inhibited by a dominant negative mutant of PI 3-kinase (Δp85) in a similar dose-dependent manner, suggesting that a small increase in the activity of Akt is not sufficient to fully inhibit PEPCK gene expression. Therefore, the simplest explanation of the present results is that a molecule distinct from Akt is capable of transmitting signals to PEPCK gene transcription, whereas activated Akt is sufficient to inhibit the expression of this gene under certain conditions.We have shown that Akt-AA almost completely abolished the stimulation of transfected Akt1 and endogenous Akt2 activity by insulin. Because antibodies against Akt3 did not precipitate insulin-stimulated kinase activity from HL1C cells, this isoform of Akt may not be expressed in these cells. Furthermore, we have shown that Akt-AA inhibited insulin-induced endogenous Akt activity precipitated with antibodies that recognize all three known isoforms of Akt with apparent similar efficiency (17Kitamura T. Ogawa W. Sakaue H. Hino Y. Kuroda S. Takata M. Matsumoto M. Maeda T. Konishi H. Kikkawa U. Kasuga M. Mol. Cell. Biol. 1998; 18: 3708-3717Crossref PubMed Scopus (295) Google Scholar). However, Akt translocates to the plasma membrane fraction or GLUT4-containing vesicles in response to extracellular stimuli (30Cron P. Cohen P. Lucocq J.M. Hemmings B.A. J. Biol. Chem. 1997; 272: 31515-31524Abstract Full Text Full Text PDF PubMed Scopus (895) Google Scholar, 31Calera M.R. Martinez C. Liu H. El Jack A.K. Birnbaum M.J. Pilch P.F. J. Biol. Chem. 1998; 273: 7201-7204Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar), suggesting that translocation of Akt to a specific intracellular compartment is important for its activation and signal transmission. Because we assayed Akt activity in immunoprecipitates prepared from total cell lysates, it is possible that this assay does not completely reflect the activity of Akt in a specific cellular compartment. We therefore cannot exclude the possibility that an increase of Akt activity in a certain fraction of the cell may be sufficient to transmit signals to the PEPCK gene promotor. It is also possible that an unidentified isoform of Akt that is resistant to Akt-AA is present in the cells studied and responsible for the regulation of PEPCK gene transcription.Liao et al. (15Liao J. Barthel A. Nakatani K. Roth R.A. J. Biol. Chem. 1998; 273: 27320-27324Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) showed that insulin-induced inhibition of PEPCK gene transcription is partially blocked in H4IIE cells that stably express Akt-AA (15Liao J. Barthel A. Nakatani K. Roth R.A. J. Biol. Chem. 1998; 273: 27320-27324Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). However, these investigators did not observe the inhibition of insulin-induced activation of endogenous Akt by this mutant. The reason for this apparent discrepancy between their results and ours is not clear. The difference could be related to the methods used to monitor PEPCK mRNA expression. Whereas we assayed transcription by assessing the activity of a CAT reporter gene fused to the promoter region of the PEPCK gene, Liao et al. (15Liao J. Barthel A. Nakatani K. Roth R.A. J. Biol. Chem. 1998; 273: 27320-27324Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) measured PEPCK mRNA by primer extension analysis. The latter technique cannot discriminate between effects on gene transcription and mRNA stability, and insulin is known to affect PEPCK mRNA stability (2O'Brien R.M. Granner D.K. Biochem. J. 1991; 278: 609-619Crossref PubMed Scopus (246) Google Scholar).We and others have shown that a kinase-defective mutant of Akt in which the Lys179 in the kinase domain is replaced by aspartate does not inhibit insulin-induced Akt activation (17Kitamura T. Ogawa W. Sakaue H. Hino Y. Kuroda S. Takata M. Matsumoto M. Maeda T. Konishi H. Kikkawa U. Kasuga M. Mol. Cell. Biol. 1998; 18: 3708-3717Crossref PubMed Scopus (295) Google Scholar, 25van Weeren P.C. de Bruyn K.M.T. de Vries-Smits A.M.M. van Lint J. Burgering B.M.T. J. Biol. Chem. 1998; 273: 13150-13156Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). Nonetheless, this and similar Akt mutants do inhibit certain biological actions of insulin, including the phosphorylation of 4E-BP1 (32Gingras A.C. Kennedy S.G. O'Leary M.A. Sonenberg N. Hay N. Genes Dev. 1998; 12: 502-513Crossref PubMed Scopus (722) Google Scholar), the activation of glycogen synthase (29Takata M. Ogawa W. Kitamura T. Hino Y. Kuroda S. Kotani K. Klip A. Gingras A.C. Sonenberg N. Kasuga J. Biol. Chem. 1999; 274: 20611-20618Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), and phosphorylation and activation of PDE3B.2 Because Bcl-XL/Bcl-2-associated death factor and glycogen synthase kinase-3β, putative in vivosubstrates of Akt, associate with Akt in intact cells (25van Weeren P.C. de Bruyn K.M.T. de Vries-Smits A.M.M. van Lint J. Burgering B.M.T. J. Biol. Chem. 1998; 273: 13150-13156Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 33Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1998; 91: 231-241Abstract Full Text Full Text PDF Scopus (4914) Google Scholar), it is possible that kinase-defective mutants of Akt containing substitutions at Lys179 block signaling downstream of Akt by competing with the endogenous enzyme for its substrates. However, in the present study, infection of HL1C cells with AxCAAkt-K179D did not prevent the inhibition of PEPCK gene transcription by insulin, which is consistent with the results of a previous study (9Agati J.M. Yeagley D. Quinn P.G. J. Biol. Chem. 1998; 273: 18751-18759Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). These observations also support the hypothesis that inhibition of Akt signaling is not sufficient to prevent insulin suppression of PEPCK gene transcription.Atypical isoforms of PKC are also thought to act downstream of PI 3-kinase (18Kotani K. Ogawa W. Matsumoto M. Kitamura T. Sakaue H. Hino Y. Miyake K. Sano W. Akimoto K. Ohno S. Kusuga M. Mol. Cell. Biol. 1998; 18: 6971-6982Crossref PubMed Google Scholar, 34Akimoto K. Takahashi R. Moriya S. Nishioka N. Takayanagi J. Kimura K. Fukui Y. Osada S.-I. Mizuno K. Hirai S.-I. Kazlauskas A. Ohno S. EMBO J. 1996; 15: 788-798Crossref PubMed Scopus (257) Google Scholar, 35Bandyopadhyay G. Standaert M.L. Zhao L. Yu B. Avignon A. Galloway L. Karnam P. Moscat J. Farese R.V. J. Biol. Chem. 1997; 272: 2551-2558Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). We have previously shown that expression of λΔNKD markedly inhibits stimulation of both PKCλ activity and glucose transport in 3T3-L1 adipocytes by insulin (18Kotani K. Ogawa W. Matsumoto M. Kitamura T. Sakaue H. Hino Y. Miyake K. Sano W. Akimoto K. Ohno S. Kusuga M. Mol. Cell. Biol. 1998; 18: 6971-6982Crossref PubMed Google Scholar). We have now shown that PKCλ, but not PKCζ, is expressed in HL1C cells and that λΔNKD does not affect the inhibition of PEPCK gene transcription by insulin, although this mutant almost completely inhibited insulin-induced activation of PKCλ. Atypical PKC has been proposed to participate in the regulation of the mitogen-activated protein kinase-signaling cascade (36Berra E. D'az-Meco M.T. Lozano J. Frutos S. Municho M.M. Sánchez P. Sanz L. Moscat J. EMBO J. 1995; 14: 6157-6163Crossref PubMed Scopus (252) Google Scholar, 37Schonwasser D.C. Marais R.M. Marshall C.J. Parker P.J. Mol. Cell. Biol. 1998; 18: 790-798Crossref PubMed Scopus (674) Google Scholar). In this regard, we have recently shown that expression of λΔNKD inhibits the insulin-induced activation of mitogen-activated protein kinase in various cells, including HL1C cells. 3Matsumoto, M., Ogawa, W., Kotani, K., and Kasuga, M., manuscript in preparation. These results, together with our observation that λΔPD, a constitutively active mutant of PKCλ, does not mimic the effect of insulin on PEPCK gene transcription, indicate that PKCλ does not participate in this effect of insulin.The small GTPase Rac, another downstream effector of PI 3-kinase, mediates insulin-induced formation of lamellipodia (38Tapon N. Hall A. Curr. Opin. Cell Biol. 1997; 9: 86-92Crossref PubMed Scopus (690) Google Scholar). Overexpression of a dominant negative mutant of Rac (Rac17N) did not prevent the inhibitory effect of insulin on PEPCK gene transcription, suggesting that the Rac pathway does not contribute to this action of insulin. Because insulin does not induce formation of lamellipodia in HL1C cells, we do not know whether the level of expression of Rac17N achieved in the present study is sufficient to prevent signaling through endogenous Rac. However, given that insulin-induced formation of lamellipodia is completely blocked in Chinese hamster ovary cells, KB cells, and 3T3-L1 adipocytes by expression of Rac17N at levels similar to or lower than that achieved in the present study (data not shown), it is likely that the concentration of Rac17N achieved in HL1C cells was sufficient to inhibit the endogenous protein.In summary, we have shown that a constitutively active mutant of PI 3-kinase inhibits the dexamethasone-cAMP-induced stimulation of PEPCK gene transcription and that a dominant negative mutant of this lipid kinase blocks insulin-induced inhibition of PEPCK gene expression. However, dominant negative mutants of Akt, PKCλ, and Rac did not affect the ability of insulin to inhibit transcription of the PEPCK gene. Our data suggest that a downstream effector of PI 3-kinase distinct from Akt, PKCλ, and Rac mediates the effect of insulin on PEPCK gene transcription. Given that various additional enzymes, including PKCε (39Moriya S. Kazlauskas A. Akimoto K. Hirai S. Mizuno K. Takenawa T. Fukui Y. Watanabe Y. Ozaki S. Ohno S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 151-155Crossref PubMed Scopus (167) Google Scholar), p21-activated kinase (40Tsakiridis T. Taha C. Grinstein S. Klip A. J. Biol. Chem. 1996; 271: 19664-19667Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), and integrin-linked kinase (41Delcommenne M. Tan C. Gray V. Rue L. Woodget J. Dedhar S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11211-11216Crossref PubMed Scopus (941) Google Scholar), have been shown to act downstream of PI 3-kinase in insulin-induced or other growth factor-induced signaling, the effects of specific inhibition of such effectors on PEPCK gene transcription warrant investigation. The primary role of insulin is to control the plasma glucose concentration by stimulating glucose transport into muscle and adipose cells as well as by reducing glucose output from the liver (1Kruszynska Y.T. Olefsky J.M. J. Invest. Med. 1996; 44: 413-428PubMed Google Scholar). These actions of insulin are mediated by activation of effectors, such as glucose transporters and glycogen synthase, or by regulation of the amount of specific protein participants in metabolic pathways (1Kruszynska Y.T. Olefsky J.M. J. Invest. Med. 1996; 44: 413-428PubMed Google Scholar, 2O'Brien R.M. Granner D.K. Biochem. J. 1991; 278: 609-619Crossref PubMed Scopus (246) Google Scholar, 3White M.F. Diabetologia. 1997; 40 Suppl. 2: 2-17Crossref PubMed Scopus (460) Google Scholar, 4Holman G.D. Kasuga M. Diabetologia. 1997; 40: 991-1003Crossref PubMed Scopus (187) Google Scholar). Phosphoenolpyruvate carboxykinase (PEPCK), 1The abbreviations used are: PEPCK, phosphoenolpyruvate carboxykinase; PI, phosphoinositide; PKC, protein kinase C; CAT, chloramphenicol acetyltransferase; HA, hemagglutinin; MOI, multiplicity of infection; pfu, plaque-forming unit1The abbreviations used are: PEPCK, phosphoenolpyruvate carboxykinase; PI, phosphoinositide; PKC, protein kinase C; CAT, chloramphenicol acetyltransferase; HA, hemagglutinin; MOI, multiplicity of infection; pfu, plaque-forming unit a rate controlling enzyme of gluconeogenesis, is one such protein whose expression is regulated by insulin (2O'Brien R.M. Granner D.K. Biochem. J. 1991; 278: 609-619Crossref PubMed Scopus (246) Google Scholar). Insulin inhibits gluconeogenesis in the liver; thus, in the absence of this effect of insulin, as in diabetes mellitus or long term starvation, gluconeogenesis is increased (1Kruszynska Y.T. Olefsky J.M. J. Invest. Med. 1996; 44: 413-428PubMed Google Scholar). Transcription of the PEPCK gene is increased by various hormonal agents including glucocorticoids and glucagon (or its second messenger, cAMP), and insulin inhibits PEPCK gene transcription induced by these stimuli (2O'Brien R.M. Granner D.K. Biochem. J. 1991; 278: 609-619Crossref PubMed Scopus (246) Google Scholar). Given that PEPCK is not known to be subject to allosteric regulation, the inhibition of gluconeogenesis by insulin in vivo is probably due to the insulin-induced decrease in the amount of PEPCK protein. Moreover, the observations that PEPCK gene expression in the liver is increased in several animal models of diabetes (5Hofmann C. Lorenz K. Williams D. Palazuk B.J. Colca J.R. Metabolism. 1995; 44: 384-389Abstract Full Text PDF PubMed Scopus (25) Google Scholar) and that transgenic animals overexpressing the PEPCK gene develop a diabetic phenotype (6Valera A. Pujol A. Pelegrin M. Bosch F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9151-9154Crossref PubMed Scopus (252) Google Scholar, 7Rosella G. Zajac J.D. Baker L. Kaczmarczyk S.J. Andrikopoulos S. Adams T.E. Proietto J. Mol. Endocrinol. 1995; 9: 1396-1404PubMed Google Scholar) also indicate the importance of this enzyme in glucose homeostasis in vivo. Despite the recent progress in our knowledge of insulin signal transduction, the mechanism by which PEPCK gene transcription is regulated remains unclear. The Ras and mitogen-activated protein kinase signaling cascade contributes to the regulation of the expression of various genes by insulin (8Davis R.J. Mol. Reprod. Dev. 1995; 42: 459-467Crossref PubMed Scopus (382) Google Scholar). A constitutively active mutant of Ras was shown to inhibit transcription of the PEPCK gene induced by cAMP-depend
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